KR20230125042A - Anti-TSLP nanoantibodies and applications thereof - Google Patents

Abstract

The present invention provides anti-TSLP nanoantibodies and applications thereof. The present invention provides anti-TSLP nanoantibodies, and the present invention provides a coding sequence encoding the nanoantibodies, a corresponding expression vector and a host cell capable of expressing the nanoantibodies, and a method for producing the nanoantibodies of the present invention. provide more The nanoantibody of the present invention has good TSLP/TSLPR blocking activity; The nanoantibody of the present invention is expressed by Pichia pastoris, and its expression yield in a fermentation tank can reach 17-23g/L.

Description

Anti-TSLP nanoantibodies and applications thereof

The present invention relates to the field of biomedicine or biopharmaceutical technology, and more particularly to anti-TSLP nanoantibodies and applications thereof.

In recent years, the treatment pathway for moderate to severe asthma has focused on the response to control Th2 cells. Most antibody drugs used to treat severe asthma target the Th2 pathway, including IgE (omalizumab), IL5 (mepolizumab, relizumab), and IL5R (benralizumab). ), IL4R (Dupilumab), and the like. All of these drugs have a good control effect on hypereosinophilic asthma, and all are treated by subcutaneous or intravenous injection. About one-third of patients with severe asthma are not characterized by activation of Th2 inflammatory pathways, and currently there are no biologic treatment options for patients with uncontrolled non-Th2-based diseases in established standard nursing care, so the development of new treatment pathways is urgently needed. do.

Thymic stromal lymphopoietin (TSLP) is a short-chain cytokine with a four helix-bundle fold structure and belongs to the IL-2 cytokine family. TSLP is an epithelial cytokine produced in response to pro-inflammatory stimuli (eg, pulmonary allergens, viruses and other pathogens) and plays an important role in the development and persistence of airway inflammation. TSLP induces the release of downstream Th2 cytokines, including IL-4, IL-5 and IL-13, resulting in inflammation and asthmatic symptoms. TSLP can also activate various cell types associated with non-Th2 induced inflammation. Thus, the early upstream activity of TSLP in the inflammatory cascade response was identified as a potential target in a broad population of asthma patients. Blocking TSLP prevents immune cells from releasing pro-inflammatory cytokines, preventing asthma exacerbations and improving asthma control and related diseases such as chronic obstructive pulmonary disease.

Nanobody (Nb), that is, heavy-chain nanoantibody (variable domain of heavy chain of heavy-chain antibody, VHH) - camels naturally have a heavy-chain antibody (HCAb) that does not have a light chain, and its variable The nanoantibody obtained by cloning the region is composed of only one heavy chain variable region, and is the smallest unit that is stable and capable of binding to an antigen with perfect functions that can be obtained today. Nanoantibodies have characteristics such as high stability, good water solubility, simple humanization, high targetability, and strong penetrability, and exert great functions beyond imagination in immunological experiments, diagnosis, and treatment. Nanoantibodies are gradually becoming a new force in next-generation antibody therapy.

Therefore, it is urgent in this field to develop anti-TSLP nanoantibodies having good blocking activity, good clinical efficacy, and easy production.

An object of the present invention is to provide anti-TSLP nanoantibodies having good blocking activity, good clinical efficacy, and easy production.

A first aspect of the present invention provides an anti-TSLP nanoantibody, wherein the complementarity determining region (CDR) of the VHH chain in the nanoantibody comprises:

(1) CDR1 represented by SEQ ID NO: 1, CDR2 represented by SEQ ID NO: 2, and CDR3 represented by SEQ ID NO: 3;

(2) CDR1 represented by SEQ ID NO: 14, CDR2 represented by SEQ ID NO: 15, and CDR3 represented by SEQ ID NO: 16; and

(3) at least one selected from the group consisting of CDR1 represented by SEQ ID NO: 27, CDR2 represented by SEQ ID NO: 28, and CDR3 represented by SEQ ID NO: 29;

In another preferred embodiment, the CDR1, CDR2 and CDR3 are separated by the frame regions (FR1, FR2, FR3 and FR4) of the VHH chain.

In another preferred example, the VHH chain further comprises a frame region (FR), the frame region (FR),

(1) FR1 represented by SEQ ID NO: 4, FR2 represented by SEQ ID NO: 5, FR3 represented by SEQ ID NO: 6, and FR4 represented by SEQ ID NO: 7;

(2) FR1 represented by SEQ ID NO: 10, FR2 represented by SEQ ID NO: 5, FR3 represented by SEQ ID NO: 6, and FR4 represented by SEQ ID NO: 11;

(3) FR1 represented by SEQ ID NO: 17, FR2 represented by SEQ ID NO: 18, FR3 represented by SEQ ID NO: 19, and FR4 represented by SEQ ID NO: 20;

(4) FR1 represented by SEQ ID NO: 23, FR2 represented by SEQ ID NO: 18, FR3 represented by SEQ ID NO: 19, and FR4 represented by SEQ ID NO: 24;

(5) FR1 represented by SEQ ID NO: 30, FR2 represented by SEQ ID NO: 31, FR3 represented by SEQ ID NO: 32, and FR4 represented by SEQ ID NO: 33; and

(6) at least one selected from the group consisting of FR1 represented by SEQ ID NO: 36, FR2 represented by SEQ ID NO: 31, FR3 represented by SEQ ID NO: 32, and FR4 represented by SEQ ID NO: 37;

In another preferred embodiment, the CDR region of the nanoantibody VHH chain is at least 80%, preferably at least 90%, more preferably at least 90% of any one of SEQ ID NOs: 1-3, SEQ ID NOs: 14-16, and SEQ ID NOs: 27-29 amino acid sequences that have at least 95%, and even more preferably at least 99%, sequence similarity.

In another preferred embodiment, the amino acid sequence of the CDR region of the nanoantibody VHH chain has one or more amino acid substitutions, preferably conserved, compared to any one of SEQ ID NOs: 1-3, SEQ ID NOs: 14-16, and SEQ ID NOs: 27-29. Including amino acid substitutions.

In another preferred embodiment, the amino acid sequence of any one of the above amino acid sequences optionally adds or deletes at least one (eg 1-3, relatively preferably 1-2, more preferably 1) amino acids. , a derivative sequence that has undergone modification and/or substitution and has the function of specifically binding to TSLP.

In another preferred example, the nanoantibody can specifically bind to TSLP.

In another preferred example, the nanoantibody can effectively block the interaction between TSLP and TSLPR.

In another preferred embodiment, the TSLP is a human or non-human mammalian TSLP.

In another preferred embodiment, the TSLP is a human, mouse, rat or non-human primate (eg monkey) TSLP.

In another preferred embodiment, the nanoantibodies include humanized antibodies, camel antibodies, and chimeric antibodies.

In another preferred embodiment, the amino acid sequence of the VHH chain of the nanoantibody is selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 34, SEQ ID NO: 38, or a combination thereof.

In another preferred embodiment, the anti-TSLP nanoantibodies include monomers, divalents (bivalent antibodies), tetravalents (tetravalent antibodies), and/or multimers (multivalent antibodies).

In another preferred embodiment, the anti-TSLP nanoantibody includes at least one VHH chain having an amino acid sequence represented by SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 34 or SEQ ID NO: 38.

In another preferred embodiment, the anti-TSLP nanoantibody includes two VHH chains having amino acid sequences represented by SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 34, SEQ ID NO: 38.

In another preferred embodiment, the VHH chains are linked through a linking peptide.

In another preferred embodiment, the connecting peptide is selected from the sequence of (G _a S _b ) _x , wherein a, b, x=0 or 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 (Comparatively preferably, a = 4, b = 1, x = 4).

In another preferred embodiment, the sequence of the linking peptide is GGGGSGGGGSGGGGSGGGGS.

A second aspect of the present invention provides an anti-TSLP antibody, which is an antibody against a TSLP epitope, and has the anti-TSLP nanoantibody according to the first aspect of the present invention.

In another preferred embodiment, the anti-TSLP antibody includes one or more anti-TSLP nanoantibodies.

In another preferred embodiment, the anti-TSLP antibody comprises monomers, divalents (bivalent antibodies), tetravalents (tetravalent antibodies), and/or multimers (multivalent antibodies).

In another preferred embodiment, the anti-TSLP antibody comprises one or more VHH chains having an amino acid sequence represented by SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 34 or SEQ ID NO: 38.

In another preferred embodiment, the anti-TSLP antibody comprises two VHH chains having an amino acid sequence represented by SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 34 or SEQ ID NO: 38.

In another preferred example, the antibody can specifically bind to TSLP.

In another preferred embodiment, the antibody can specifically target a TSLP protein with a precise spatial structure.

In another preferred embodiment, the antibody can effectively block the interaction between TSLP and TSLPR.

In another preferred embodiment, the affinity (KD value) of the antibody for TSLP is less than 3.77 nM.

In another preferred embodiment, the antibody has good TSLP/TSLPR blocking activity, and the blocking activity is significantly better than the control antibody Tezepelumab, wherein the control antibody Tezepelumab was obtained from AstraZeneca or Amgen.

In another preferred embodiment, the antibody can effectively inhibit the proliferation of BaF3/TSLPR-IL7R cells, and its inhibitory activity is superior to that of the control antibody Tezepelumab.

In another preferred embodiment, the antibody is a nanoantibody.

A third aspect of the present invention provides a polynucleotide, wherein the polynucleotide is a protein selected from the group consisting of the anti-TSLP nanoantibody according to the first aspect of the present invention or the anti-TSLP antibody according to the second aspect of the present invention to code

In another preferred embodiment, the polynucleotides are in combination form.

In another preferred embodiment, the polynucleotide sequence includes one or more of the sequences represented by SEQ ID NO: 9, 13, 22, 26, 35 or 39.

In another preferred embodiment, the polynucleotide comprises RNA, DNA or cDNA.

A fourth aspect of the present invention provides an expression vector containing the polynucleotide according to the third aspect of the present invention.

In another preferred embodiment, the expression vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, other gene delivery systems, or combinations thereof.

In another preferred embodiment, the expression vector includes a viral vector such as a lentivirus, adenovirus, AAV virus, or retrovirus.

A fifth aspect of the present invention provides a host cell, wherein the host cell contains the expression vector according to the fourth aspect of the present invention or has the polynucleotide according to the third aspect of the present invention integrated into its genome.

In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cell, mammalian cell, phage, or combinations thereof.

In another preferred embodiment, the prokaryotic cell is selected from the group consisting of Escherichia coli, Bacillus subtilis, Lactobacillus, Streptomyces, Proteus mirabilis, or a combination thereof.

In another preferred embodiment, the eukaryotic cell is selected from the group consisting of Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces, Trichoderma, or combinations thereof.

In another preferred embodiment, the host cell is Pichia pastoris.

A sixth aspect of the present invention provides a method for generating an anti-TSLP nanoantibody, the method comprising:

(a) obtaining a culture containing the anti-TSLP nanoantibody by culturing the host cell according to the fifth aspect of the present invention under conditions suitable for producing the nanoantibody;

(b) isolating or recovering the anti-TSLP nanoantibody from the culture; and

(c) optionally purifying and/or modifying the anti-TSLP nanoantibody obtained in step (b);

A seventh aspect of the present invention provides an immunoconjugate, wherein the immunoconjugate comprises:

(a) the anti-TSLP nanoantibody according to the first aspect of the present invention, or the anti-TSLP antibody according to the second aspect of the present invention; and

(b) contains a junction moiety selected from the group consisting of detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, gold nanoparticles/nanorods, magnetic nanoparticles, viral coat proteins or VLPs, or combinations thereof. do.

In another preferred example, the radionuclide includes the following isotopes,

(i) diagnostic isotopes, the diagnostic isotopes are Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr- 89, C-11, Lu-177, Re-188, or a combination thereof; and/or

(ii) therapeutic isotopes, the therapeutic isotopes being Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P- 32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133, Yb-169, Yb-177, or these It is selected from the group consisting of combinations of.

In another preferred embodiment, the conjugation moiety is a drug or toxin.

In another preferred embodiment, the drug is a cytotoxic drug.

In another preferred embodiment, the cytotoxic drug is an anti-tubulin drug, a DNA minor groove binding reagent, a DNA replication inhibitor, an alkylating agent, an antibiotic, a folic acid antagonist, an antimetabolite, a chemosensitizer, a topoisomerase inhibitor, a vinca alkaloid, or any of these It is selected from the group consisting of combinations.

In another preferred embodiment, examples of particularly useful cytotoxic drugs include DNA minor groove binding reagents, DNA alkylating agents, tubulin inhibitors, typical cytotoxic drugs include auristatins, camptothecins, Duokarma Containing isins/duocarmycins, etoposide, maytansines and maytansinoids (eg DM1 and DM4), taxanes, benzodiazepines or benzodiazepines benzodiazepine containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines and oxazolidinobenzodiazepines), vinca alkaloids, or combinations thereof includes

In another preferred embodiment, the toxin is an auristatin (e.g., auristatin E, auristatin F, MMAE and MMAF), aureomycin, maytansinol, ricin, lysine Chain A, combretastatin, duocarmycin, multicarcinoid Lalastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin , cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxide dihydroxyanthraxindione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE)A, PE40, abrin, abrin A chain, syringa root Toxin A chain, α-Sarcina, gelonin, mitogellin, retstrictocin, phenomycin, enonomycin, curisin (curicin), crotonin, calicheamicin, Sapaonaria officinalis inhibitor, glucocorticoid, or a combination thereof.

In another preferred embodiment, the junction is a detectable marker.

In another preferred embodiment, the junction is a fluorescent or luminescent marker, a radioactive marker, an MRI (magnetic resonance imaging) or CT (electronic computed tomography) contrast agent, or an enzyme capable of producing a detectable product, a radionuclide, a biological Toxins, cytokines (e.g. IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, viral particles, liposomes, magnetic nanoparticles, prodrug-activated enzymes (e.g. DT-diaphora) enzyme (DTD) or biphenylhydrolase-like protein (BPHL)) or nanoparticles in any form.

An eighth aspect of the present invention provides a multispecific antibody comprising the anti-TSLP nanoantibody according to the first aspect of the present invention or the anti-TSLP antibody according to the second aspect of the present invention.

In another preferred embodiment, the multispecific antibody further comprises an Fc portion of an antibody.

A ninth aspect of the present invention provides a recombinant protein, wherein the recombinant protein comprises:

(i) the anti-TSLP nanoantibody according to the first aspect of the present invention, or the anti-TSLP antibody according to the second aspect of the present invention; and

(ii) has an optional tag sequence to aid expression and/or purification.

In another preferred embodiment, the tag sequence includes an Fc tag, an HA tag and a 6His tag.

In another preferred embodiment, the recombinant protein specifically binds to the TSLP protein.

A tenth aspect of the present invention provides a pharmaceutical composition, wherein the pharmaceutical composition comprises:

(i) the anti-TSLP nanoantibody according to the first aspect of the present invention, or the anti-TSLP antibody according to the second aspect of the present invention, or the immunoconjugate according to the seventh aspect of the present invention or the eighth aspect of the present invention a multispecific antibody according to the present invention or a recombinant protein according to the ninth aspect of the present invention; and

(ii) contains a pharmaceutically acceptable carrier.

In another preferred embodiment, the conjugation portion of the immunoconjugate is a drug, toxin, and/or therapeutic isotope.

In another preferred embodiment, the pharmaceutical composition further contains other drugs for treating immune system diseases or tumor diseases.

In another preferred embodiment, the other drug for treating the immune system disease or tumor disease is budesonide, fluticasone, beclomethasone, mometasone furoate, albuterol (albuterol), theophylline, formoterol, tiotropium, sulfasalazine, methotrexate, cyclophosphamide, fluorouracil, bleomycin (bleomycin), anastrozole, or a combination thereof.

In another preferred embodiment, the pharmaceutical composition is used for the preparation of a drug for the prevention and/or treatment of a TSLP-related disease or condition.

In another preferred embodiment, the TSLP-related disease or condition includes an immune system disease or a neoplastic disease.

In another preferred embodiment, the immune system disease is asthma, atopic dermatitis, chronic obstructive pulmonary disease (COPD), allergic conjunctivitis, food allergy, ulcerative colitis, Crohn's disease, rhinitis, ankylosing spondylitis, systemic lupus erythematosus, rheumatoid arthritis , hypersensitivity pneumonitis, allergic granulomatous vasculitis, nasal polyposis, or a combination thereof.

In another preferred embodiment, the tumor disease is selected from the group consisting of breast cancer, pancreatic cancer, cervical cancer, multiple myeloma, colorectal cancer, lung cancer, thyroid cancer, ovarian cancer, liver cancer, or combinations thereof.

An eleventh aspect of the present invention is directed to (a) preparing a drug for preventing and/or treating a TSLP-related disease; and/or (b) the anti-TSLP nanoantibody according to the first aspect of the present invention, the anti-TSLP antibody according to the second aspect of the present invention, the seventh aspect of the present invention for preparing a TSLP detection reagent, detection plate or kit or the multispecific antibody according to the eighth aspect of the present invention or the recombinant protein according to the ninth aspect of the present invention or the pharmaceutical composition according to the tenth aspect of the present invention.

In another preferred embodiment, the TSLP-related disease or condition includes an immune system disease or a neoplastic disease.

In another preferred embodiment, the immune system disease is asthma, atopic dermatitis, chronic obstructive pulmonary disease (COPD), allergic conjunctivitis, food allergy, ulcerative colitis, Crohn's disease, rhinitis, ankylosing spondylitis, systemic lupus erythematosus, rheumatoid arthritis , hypersensitivity pneumonitis, allergic granulomatous vasculitis, nasal polyposis, or a combination thereof.

In another preferred embodiment, the tumor disease is selected from the group consisting of breast cancer, pancreatic cancer, cervical cancer, multiple myeloma, colorectal cancer, lung cancer, thyroid cancer, ovarian cancer, liver cancer, or combinations thereof.

In another preferred embodiment, the TSLP is human TSLP.

In another preferred embodiment, the reagent is a diagnostic reagent.

In another preferred embodiment, the diagnostic agent is a contrast agent.

In another preferred embodiment, the reagent is used to detect a TSLP protein or fragment thereof in a sample.

In another preferred embodiment, the detection includes flow cytometry detection, cellular immunofluorescence detection.

In another preferred embodiment, the use is diagnostic and/or non-diagnostic and/or therapeutic and/or non-therapeutic.

A twelfth aspect of the present invention provides a method for detecting TSLP protein in a sample, the method comprising:

(1) the sample is treated with the anti-TSLP nanoantibody according to the first aspect of the present invention, or the anti-TSLP antibody according to the second aspect of the present invention, or the immunoconjugate according to the seventh aspect of the present invention or the eighth aspect of the present invention contacting the multispecific antibody according to the aspect or the recombinant protein according to the ninth aspect of the present invention; and

(2) detecting whether an antigen-antibody complex is formed, indicating that the TSLP protein is present in the sample when the complex is formed;

In another preferred embodiment, the method is a non-diagnostic and non-therapeutic method.

A thirteenth aspect of the present invention provides a TSLP protein detection reagent, wherein the detection reagent comprises:

(i) the anti-TSLP nanoantibody according to the first aspect of the present invention, or the anti-TSLP antibody according to the second aspect of the present invention, or the immunoconjugate according to the seventh aspect of the present invention or the eighth aspect of the present invention a multispecific antibody according to the present invention or a recombinant protein according to the ninth aspect of the present invention; and

(ii) a detectably acceptable carrier.

In another preferred embodiment, the conjugation portion of the immunoconjugate is a diagnostic isotope.

In another preferred embodiment, the detectably acceptable carrier is a non-toxic, inert aqueous carrier medium.

In another preferred embodiment, the detection reagent is one or more reagents selected from the group consisting of isotope tracers, contrast agents, flow detection reagents, cell immunofluorescence detection reagents, magnetic nanoparticles and imaging agents.

In another preferred embodiment, the detection reagent is used for in vivo detection.

In another preferred embodiment, the formulation of the detection reagent is a liquid or powder (eg, water formulation, injection, lyophilized powder, tablet, oral formulation, aerosol).

A fourteenth aspect of the present invention provides a kit for detecting a TSLP protein, the kit comprising the immunoconjugate according to the seventh aspect of the present invention or a detection reagent and instructions according to the thirteenth aspect of the present invention.

In another preferred embodiment, according to the above instructions, the kit is used to non-invasively detect the expression of TSLP in a subject.

A fifteenth aspect of the present invention provides use of the immunoconjugate according to the seventh aspect of the present invention for preparing a contrast agent for detecting TSLP protein in vivo.

In another preferred embodiment, the detection is used for diagnosis or prognosis of a TSLP-related disease or condition.

The sixteenth aspect of the present invention provides a method for treating a disease, the method comprising administering the anti-TSLP nanoantibody according to the first aspect of the present invention, the anti-TSLP antibody according to the second aspect of the present invention, to a subject in need thereof. administering the immunoconjugate according to the seventh aspect or the multispecific antibody according to the eighth aspect of the present invention or the recombinant protein according to the ninth aspect of the present invention or the pharmaceutical composition according to the tenth aspect of the present invention.

In another preferred embodiment, the subject includes a human or non-human mammal.

In another preferred embodiment, the non-human mammal includes rodents (eg, mice, rabbits) and non-human primates (eg, monkeys).

It should be understood that within the scope of the present invention, each of the above technical features of the present invention and each technical feature specifically described below (eg, in the embodiments) can be combined with each other to form a new or desirable technical solution. Due to the limitation of the width, it is not repeated here one by one.

Figures 1a and 1b show the results of detection of blocking activity by flow cytometry for candidate antibodies of 31 weeks. As a result, the blocking activity of the 14-week antibody among the 31-week candidate antibodies is significantly superior to that of the control antibody Tezepelumab.
Figure 2 is a detection result of the binding kinetics of the 14-week blocking TSLP nanoantibody.
Figure 3 is the SDS-PAGE result of the shake flask expression supernatants of bivalent single domain antibodies Bi-HuNb5-31, Bi-HuNb7-54, and Bi-HuNb10-63 in Pichia pastoris, the yields were 475 ug/mL, respectively. 310ug/mL, 510ug/mL.
Figure 4 is the result of detecting the blocking activity of the humanized bivalent antibody by ELISA. As a result, the blocking activity of the 3-week humanized bivalent antibody is significantly superior to that of the control antibody Tezepelumab.
5 shows the results showing the inhibitory effect of the humanized bivalent antibody on the proliferation of BaF3/TSLPR-IL7R cells. As a result, the inhibitory effect of the two humanized antibodies on the pSTAT5 signaling pathway in BaF3/TSLPR-IL7R cells was superior to that of the control antibody Tezepelumab.

An anti-TSLP nanoantibody was unexpectedly discovered for the first time, and experimental results showed that the nanoantibody of the present invention can effectively block the interaction between TSLP and TSLPR, and the blocking activity is significantly superior to that of the control antibody Tezepelumab; The nanoantibody of the present invention can effectively inhibit the proliferation of Baf3/TSLPR-IL7R cells, and its inhibitory activity is superior to that of the control antibody Tezepelumab; In Pichia pastoris, the fermentation tank expression level of the nanoantibody of the present invention can reach 17-23 g/L, which is significantly higher than the industry level. On this basis, the present inventors completed the present invention.

Terms

As used herein, the terms “nanoantibodies of the present invention”, “nanoantibodies of the present invention”, “anti-TSLP nanoantibodies of the present invention”, “TSLP nanoantibodies of the present invention”, “anti-TSLP nanoantibodies” ”, “TSLP nanoantibody” have the same meaning and can be used interchangeably, and all refer to nanoantibodies that specifically recognize and bind to TSLP (including human TSLP).

As used herein, the term “antibody” or “immunoglobulin” is a heterotetrameric glycoprotein of about 150,000 daltons with identical structural characteristics consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to the heavy chain by one covalent disulfide bond, and the number of disulfide bonds differs between heavy chains of the isotype of different immunoglobulins. Each heavy and light chain also has regularly spaced intra-chain disulfide bonds. At one end of each heavy chain is a variable region (VH) followed by a plurality of constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; The constant region of the light chain and the first constant region of the heavy chain are opposite, and the variable region of the light chain and the variable region of the heavy chain are opposite. Special amino acid residues form the interface between the variable regions of the light and heavy chains.

As used herein, the terms "single domain", "VHH", "nanobody" and "heavy chain antibody" (single domain antibody, sdAb, or nanobody) have the same meaning and are used interchangeably It can be used, refers to cloning for the variable region of an antibody heavy chain, and constructs a nanoantibody (VHH) composed of only one heavy chain variable region, which is a minimal antigen-binding fragment with full functionality. In general, first, an antibody in which the light chain and heavy chain constant regions 1 (CH1) are naturally deleted is obtained, and then the antibody heavy chain variable region is cloned to construct a nanoantibody (VHH) composed of only one heavy chain variable region.

As used herein, the term "variable" indicates that portions of the variable regions of an antibody differ somewhat in sequence that form the binding and specificity of the various specific antibodies for their particular antigen. However, variability is not evenly distributed throughout the antibody variable region. It is concentrated in three fragments of the light chain and heavy chain variable regions called complementarity determining regions (CDRs) or hypervariable regions. A relatively conserved part of the variable region is called a frame region FR. The variable regions of native heavy and light chains each contain four FR regions in approximate b-fold configuration connected by three CDRs forming linking loops, which in some cases may form partial b-fold structures. . The CDRs of each chain are brought into proximity by FR regions and together with the CDRs of the other chains form the antigen-binding site of an antibody (Kabat et al., NIH Publ. No. 91-3242, Vol.I, pp. 647-669 ( 1991)). The constant region does not directly participate in the binding of the antibody to the antigen, but exhibits different effector functions, such as, for example, participating in antibody-dependent cytotoxicity of the antibody.

As is known to those skilled in the art, immunoconjugates and fusion expression products include conjugates formed by binding drugs, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules to an antibody or fragment thereof of the invention. . The present invention further includes a cell surface marker or antigen bound to the anti-TSLP antibody or fragment thereof.

As used herein, the terms “heavy chain variable region” and “VH” are used interchangeably.

As used herein, the terms “variable region” and “complementarity determining region (CDR)” may be used interchangeably.

In a preferred embodiment of the present invention, the heavy chain variable region of the antibody includes three complementarity determining regions CDR1, CDR2, and CDR3.

In one preferred embodiment of the present invention, the heavy chain of the antibody includes the heavy chain variable region and the heavy chain constant region.

In the present invention, the terms "antibody of the present invention", "protein of the present invention", or "polypeptide of the present invention" may be used interchangeably, and all refer to a polypeptide specifically binding to the TSLP protein, for example A protein or polypeptide having a heavy chain variable region. They may or may not contain a starting methionine.

The invention further provides other proteins or fusion expression products with the antibodies of the invention. Specifically, the present invention relates to any heavy chain having a variable region comprising a variable region, provided that the variable region is identical to, or has at least 90% homology, relatively preferably at least 95% homology with the heavy chain variable region of an antibody of the present invention. Includes proteins or protein conjugates and fusion expression products (ie, immunoconjugates and fusion expression products).

In general, the antigen-binding properties of antibodies can be described by three specific regions located in the heavy chain variable region, referred to as variable regions (CDRs), which are divided into four frame regions (FRs) and the amino acid sequences of the four FRs. is relatively conservative and does not directly participate in binding reactions. These CDRs form a cyclic structure, the β folds formed through the FRs between them are close to each other in a spatial structure, and the CDRs of the heavy chain and the CDRs of the corresponding light chain constitute the antigen binding site of the antibody. The amino acid sequences of relatively identical types of antibodies can determine the amino acids that make up the FR or CDR regions.

The variable regions of the antibodies of the present invention are of interest because at least some of them are involved in antigen binding. Therefore, the present invention provides a molecule having an antibody heavy chain variable region having a CDR, as long as the CDR and the CDR identified herein have a homology of 90% or more (preferably 95% or more, and most preferably 98% or more). include

The present invention not only includes complete antibodies, but also includes fragments of antibodies having immune activity or fusion proteins formed with sequences different from those of the antibodies. Accordingly, the present invention further includes fragments, derivatives and analogues of the above antibodies.

As used herein, the terms "fragment", "derivative" and "analog" refer to a polypeptide that retains essentially the same biological function or activity as an antibody of the invention. A polypeptide fragment, derivative or analogue of the present invention may be a polypeptide in which (i) one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, such substituted amino acid residues in the genetic code. It may or may not be encoded by (ii) a polypeptide having substitutions at one or more amino acid residues, or (iii) a mature polypeptide may be converted into another compound (e.g., a polypeptide such as polyethylene glycol). (iv) a polypeptide formed by fusing additional amino acid sequences to the polypeptide sequence (e.g., a leader sequence or secretory sequence or a sequence used to purify the polypeptide; or protein sequence, or a fusion protein formed with a 6His tag). In accordance with the teachings herein, such fragments, derivatives and analogues are within the scope known to those skilled in the art.

The antibody of the present invention refers to a polypeptide having TSLP-binding activity and containing the CDR region. The term further includes variant forms of polypeptides having the same functions as the antibodies of the present invention and comprising the CDR regions. Such variant forms are deletions, insertions and/or deletions of one or more (generally 1-50, relatively preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids. substitution, and addition of one or more amino acids (generally within 20, relatively preferably within 10, more preferably within 5) to the C-terminus and/or N-terminus. For example, substitutions with amino acids that are close or similar in performance in this field generally do not alter the function of the protein. Also, adding one or more amino acids to, for example, the C-terminus and/or the N-terminus generally does not alter the function of the protein. The term further includes active fragments and active derivatives of the antibodies of the present invention.

Variant forms of the polypeptide may be homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, coated with DNA capable of hybridizing to the coding DNA of the antibody of the present invention under high or low stringency conditions. proteins, and polypeptides or proteins obtained as antisera resistant to the antibodies of the present invention.

The present invention further provides other polypeptides such as nanoantibodies or fusion proteins including fragments thereof. Except for the nearly full-length polypeptide, the present invention further includes fragments of the nanoantibody of the present invention. Generally, such fragments contain at least about 50 contiguous amino acids, relatively preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of an antibody of the invention. have

In the present invention, "conservative variants of the antibody of the present invention" are at most 10 compared to the amino acid sequence of the antibody of the present invention, relatively preferably at most 8, more preferably at most 5, most preferably at most It means that three amino acids are replaced by amino acids with similar or close properties to form a polypeptide. Most preferably, such conservative variant polypeptides are generated by amino acid substitutions according to Table A.

Table A

The present invention further provides a polynucleotide molecule encoding the antibody or fragment thereof or fusion protein thereof. A polynucleotide of the present invention may be in the form of DNA or RNA. Types of DNA include cDNA, genomic DNA or artificially synthesized DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand.

A polynucleotide encoding a mature polypeptide of the present invention includes a coding sequence encoding only the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; It includes the coding sequence of the mature polypeptide (optional additional coding sequence) and non-coding sequences.

The term “polynucleotide encoding a polypeptide” may include a polynucleotide encoding the polypeptide, and may include polynucleotides of additional coding and/or non-coding sequences.

The present invention also relates to a polynucleotide that hybridizes with said sequence and has at least 50%, relatively preferably at least 70%, more preferably at least 80% homology between the two sequences. The present invention relates to polynucleotides capable of hybridizing with the above polynucleotides of the present invention under particularly stringent conditions. In the present invention, “stringent conditions” include (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60° C.; or (2) addition of a denaturant such as 0% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42° C., etc. during hybridization; or (3) hybridization occurs only when the homology between the two sequences is at least 90% or more, more preferably 95% or more. In addition, a polypeptide encoded by a hybridizable polynucleotide has the same biological function and activity as a mature polypeptide.

The full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can generally be obtained by PCR amplification, recombination or artificial synthesis. A feasible method is to synthesize the relevant sequences by artificial synthesis, especially when the fragment length is relatively short. In general, a plurality of small fragments can be first synthesized and then ligated to obtain long-sequence fragments. In addition, a fusion protein can also be formed by fusing the coding sequence of the heavy chain with an expression tag (eg, 6His).

Once related sequences are obtained, the related sequences can be obtained collectively by recombination. It is generally obtained by cloning it into a vector, transforming it into cells, and then isolating the relevant sequences from the host cell grown in the usual way. Biological molecules (nucleic acids, proteins, etc.) involved in the present invention include biological molecules that exist in an isolated form.

Currently, DNA sequences encoding the proteins of the present invention (or fragments thereof, or derivatives thereof) can be obtained entirely through chemical synthesis. The DNA sequence can then be introduced into cells with various existing DNA molecules (or vectors, for example) known in the art. In addition, mutations may be introduced into the protein sequence of the present invention through chemical synthesis.

The present invention also relates to a vector comprising the above suitable DNA sequences and suitable promoter or control sequences. Such vectors can transform appropriate host cells to express the protein.

Host cells include prokaryotic cells such as bacterial cells; or lower eukaryotic cells such as yeast cells; or higher eukaryotic cells such as mammalian cells. Representative examples include Escherichia coli, Streptomyces; bacterial cells of Escherichia coli; fungal cells such as yeast; Drosophila S2 or Sf9 insect cells; There are animal cells such as CHO, COS7, and 293 cells.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. If the host is a prokaryotic organism such as Escherichia coli, water-soluble cells capable of uptake of DNA can be harvested after the exponential growth phase and subjected to the CaCl ₂ method using procedures well known in the art. Another method is to use MgCl ₂ . Transformation can also be performed by electroporation if desired. When the host is a eukaryote, DNA transfection methods such as calcium phosphate co-precipitation and conventional mechanical methods such as microinjection, electroporation, and liposome packaging may be used.

The obtained transformant can be cultured by a general method and expresses the polypeptide encoded by the gene of the present invention. A medium used for culture may be selected from various general media depending on the host cell used. Culture in conditions suitable for the growth of the host cell. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (eg, on-conversion or chemical induction) and the cells are cultured for a further period of time.

In the method, the recombinant polypeptide may be expressed intracellularly or in a cell membrane, or secreted extracellularly. Recombinant proteins can be separated and purified by various separation methods using physical, chemical and other properties, if necessary. These methods are well known to those skilled in the art. Examples of these methods are conventional regeneration treatment, treatment with protein precipitating agents (salting out), centrifugation, osmotic disruption, ultra treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

Antibodies of the present invention may be used alone or linked or conjugated with any combination of detectable markers (for diagnostic purposes), therapeutic agents, PK (protein kinase) modified moieties or aberrant substances.

Detectable markers for diagnostic purposes include, but are not limited to, fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (electronic computed tomography) contrast agents, or enzymes capable of producing a detectable product. .

Therapeutic agents that may be associated with or conjugated to the antibodies of the present invention include: 1. radionuclides; 2. Biological toxins; 3. Cytokines such as IL-2; 4. Gold nanoparticles/nanorods; 5. viral particles; 6. liposomes; 7. magnetic nanoparticles; 8. Prodrug activating enzymes (eg, DT-diaphorase (DTD) or biphenylhydrolase-like protein (BPHL));

Thymic stromal lymphopoietin (TSLP)

Thymic stromal lymphpoietin is a single-chain cytokine with a four-helical bundle folding structure and belongs to the IL-2 cytokine family. As a novel cytokine, TSLP is normally expressed in epithelial cells of the lung, skin and intestinal barrier surfaces. As a result of animal experiments, TSLP was highly expressed in the lungs of allergen-induced asthma model mice, whereas TSLP receptor-deficient mice had significantly reduced asthma performance, and lung-specific TSLP transgenic mice showed Th2-type inflammation and increased airway inflammation with IgE. and high reactivity. Further studies revealed that TSLP activates bone marrow-derived dendritic cells and upregulates costimulatory molecules to produce Th2-type cell chemotactic factor CCL17. Thus, TSLP is an important factor and a necessary condition for the initiation of airway allergic inflammation. It is an upstream regulator of several inflammatory pathways in various diseases including asthma and is critical for the development and persistence of airway inflammation. Studies have also shown that TSLP is also a cytokine involved in the pathogenesis of atopic dermatitis (AD). More importantly, researchers found that TSLP levels are increased in various types of tumors, and that TSLP can protect tumors from death by inducing tumor cells to express another protein called BCL-2. Therefore, TSLP is also very important for tumor survival.

Thymic stromal lymphopoietin receptor (TSLPR)

The TSLPR receptor is a type I transmembrane protein and belongs to the family of hematopoietic cytokine receptors. A functional TSLPR complex is composed of TSLPR and IL-7Ra. TSLPR is also referred to as cytokine receptor-like molecule 2 or type I cytokine receptor σ1. TSLP binds to the high-affinity heterodimeric receptor complex to initiate intracellular type 2 signal transduction, and the heterodimeric receptor complex is composed of its specific receptor TSLPR, IL-2, IL-4, IL-9 and IL-15 It has 24% homology to the consensus receptor γ chain of , and does not contain the δ-combined chain IL-2 family, and the IL-7Ra subunit (CD127) in cells co-expressing TSLPR and IL-7Rα. TSLP first binds to TSLPR and then recruits the IL-7Rα chain.

pharmaceutical composition

The present invention further provides a composition. Preferably, the composition is a pharmaceutical composition and contains the antibody or active fragment thereof or fusion protein thereof and a pharmaceutically acceptable carrier. In general, these substances can be formulated in a non-toxic, inert, pharmaceutically acceptable aqueous carrier medium, wherein the pH value can vary somewhat depending on the nature of the substance being formulated and the condition being treated, but is generally about 5 -8, relatively preferably a pH of about 6-8. The formulated pharmaceutical composition may be administered via a common route, including but not limited to intraperitoneal, intravenous, or topical administration.

The pharmaceutical composition of the present invention directly binds to a TSLP protein molecule and can be used to treat TSLP-related diseases or conditions (including immune system diseases or tumor diseases). In addition, other therapeutic agents may also be used in combination.

The pharmaceutical composition of the present invention is a safe and effective amount (eg, 0.001-99wt%, relatively preferably 0.01-90wt%, more preferably 0.1-80wt%) of the nanoantibody (or conjugate thereof) according to the present invention and Contains a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The drug formulation should be consistent with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, and is prepared by a general method, for example, using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions are prepared under aseptic conditions, such as injections and solutions. The dosage of the active ingredient is a therapeutically effective amount, for example about 10 μg/kg body weight to about 50 mg/kg body weight per day. In addition, the polypeptides of the present invention may also be used in combination with other therapeutic agents.

When using the pharmaceutical composition, a safe effective amount of the immunoconjugate is administered to the mammal, wherein the safe effective amount is usually at least about 10 μg/kg body weight, and in most cases does not exceed about 50 mg/kg body weight, relatively preferably The dosage is from about 10 μg/kg body weight to about 10 mg/kg body weight. Of course, the specific dosage should also consider factors such as the route of administration and the health condition of the patient, which are within the skill of a skilled physician.

Anti-TSLP nanoantibody

In the present invention, the amino acid sequence of the VHH chain of the anti-TSLP nanoantibody is selected from one or more of SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 34 or SEQ ID NO: 38.

In a preferred embodiment of the present invention, the anti-TSLP nanoantibodies include monomers, divalents (bivalent antibodies), tetravalents (tetravalent antibodies), and/or polyvalent antibodies (multivalent antibodies).

Typically, the anti-TSLP nanoantibody comprises two VHH chains having an amino acid sequence represented by SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 34 or SEQ ID NO: 38.

In another preferred embodiment, the VHH chains are linked via linking peptides.

In another preferred embodiment, the connecting peptide is selected from the sequence of (G _a S _b ) _x , wherein a, b, x=0 or 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 (Comparatively preferably, a = 4, b = 1, x = 4).

In another preferred embodiment, the sequence of the linking peptide is GGGGSGGGGSGGGGSGGGGS.

labeled nanoantibodies

In a preferred embodiment of the present invention, the nanoantibody has a detectable marker. More preferably, the marker is selected from the group consisting of isotopes, colloidal gold markers, color markers or fluorescent markers.

Colloidal gold marking can be performed by methods known to those skilled in the art. In a preferred solution of the present invention, nanoantibodies of TSLP are labeled with colloidal gold to obtain nanoantibodies labeled with colloidal gold.

detection method

The present invention also relates to methods for detecting TSLP proteins. The steps of the method are roughly as follows. obtaining cell and/or tissue samples; dissolving the sample into the medium; Detect the level of TSLP protein in the lysed sample.

In the detection method of the present invention, the sample used is not particularly limited, and a typical example is a cell-containing sample present in a cell preservation solution.

kit

The present invention further provides a kit comprising the antibody (or fragment thereof) or detection plate of the present invention, and in a preferred embodiment of the present invention, the kit further includes a container, instructions for use, a buffer and the like.

The present invention also provides a detection kit for detecting the level of TSLP, the kit comprising an antibody recognizing the TSLP protein, a pyrolysis medium for dissolving the sample, common reagents and buffers necessary for detection, such as various buffers, detection markers , detection substrates, and the like. The detection kit may be an in vitro diagnostic device.

Applications

As described above, the nanoantibody of the present invention has a wide range of biological and clinical application values, and its applications are involved in diagnosis and treatment of TSLP-related diseases or conditions, basic medical research, biological research, and various fields. One preferred application is clinical diagnosis and targeted therapy for TSLP.

The main advantages of the present invention include:

(a) The nanoantibody of the present invention can effectively block the interaction between TSLP and TSLPR.

(b) The nanoantibody of the present invention has stronger blocking activity than the control antibody Tezepelumab.

(c) The nanoantibody of the present invention can be expressed in Pichia pastoris, the expression output in the shake flask is relatively high, and the fermentation tank output can reach 17-23 g/L.

(d) The inhibitory effect of the nanoantibody of the present invention on the pSTAT5 signaling pathway in BaF3/TSLPR-IL7R cells is significantly superior to that of the control antibody Tezepelumab.

The present invention is further illustrated by combining specific examples below. It should be understood that these examples are intended to illustrate the invention and not to limit the scope of the invention. Experimental methods for which specific conditions are not specified in the examples below are general conditions, for example, conditions according to Sambrook et al., Molecular Clones: Laboratory Handbook (New York: Cold Spring Harbor Laboratory Press, 1989), or conditions recommended by manufacturers. follow Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

Unless otherwise specified, all materials and reagents used in the examples of the present invention are commercially available products.

Example 1 Screening of TSLP-specific nanoantibodies

After optimizing the amino acid sequence of human TSLP with a purine site mutation according to human codons, its base sequence was cloned into a pFUSE vector, transfected into HEK293F cells, and purified into human TSLP protein. After mixing high-purity protein and adjuvant, 4 renal bactrian camels were immunized once a week, and after 7 immunizations, camel peripheral blood was collected, RNA was isolated, and the VHH gene fragment was amplified, cloned into pMECS vector, and TG1 soluble cells was electrotransformed to construct a high-quality phage display nanoantibody library. As a result of the detection, the storage capacity of all four libraries reached 1x10 ⁹ CFU or more, and the fragment insertion rate was 80% or more.

TSLP-specific nanoantibodies were screened using phage display technology. After three cycles of “adsorption-washing-concentration”, specific nanoantibody phages were enriched in each library. 1200 clones were randomly selected and subjected to ELISA detection and sequencing analysis to obtain the final 312 strains of differential sequence antibodies.

Example 2 Screening of blocking TSLP nanoantibodies by flow cytometry

The nanoantibody clones having different sequences were inoculated and cultured in TB medium, induced with IPTG overnight, and the cells were pyrolyzed to collect the supernatant to detect the blocking activity. The cultured CHOZEN/TSLPR stably transfected cells were divided into 96-well plates at 3E 5 cells per well, centrifuged at 3000 rpm for 3 minutes to remove the supernatant, and the pyrolyzed solution of each antibody and TSLP-Biotin protein were added to 20 Incubated for minutes. The supernatant was discarded by centrifugation, and diluted SA-PE antibody was added and incubated at 4° C. for 20 minutes. After centrifugation again, the supernatant was discarded, 200uL PBS was added to each well to resuspend the cells, and the PE signal of the sample was detected by flow cytometry. As a result, a total of 64 candidate antibodies had the function of blocking the interaction between TSLP and TSLPR. Combined with sequencing, 31 different classes of antibodies were selected for expression and purification. See Example 4 of patent CN110144011A for purification methods.

Blocking activity was detected again by flow cytometry for the purified antibodies of the 31 weeks. Divide the cultured HEK293F/TSLPR transiently transfected cells into a 96-well plate at 5 cells of 3E per well, remove the supernatant by centrifuging at 3000 rpm for 3 minutes, remove the supernatant, and add each antibody (2x starting at 20ug/mL) in gradient dilution Gradient dilution) and TSLP-Biotin protein were added and incubated for 20 minutes. In this experiment, Tezepelumab was used as a control antibody. Subsequently, the supernatant was discarded by centrifugation, and diluted SA-PE antibody was added and incubated at 4° C. for 20 minutes. After centrifugation again, the supernatant was discarded, 200uL PBS was added to each well to resuspend the cells, and the PE signal of the sample was detected by flow cytometry. As a result, as shown in Figures 1a and 1b, the blocking activity of the antibody of 14 weeks here is significantly superior to the control antibody Tezepelumab, and the numbers of the antibodies of 14 weeks are Nb1-41, Nb1-51, Nb1-59, respectively. Nb3-18, Nb3-43, Nb5-31, Nb6-29, Nb7-54, Nb10-55, Nb10-63, Nb10-87, Nb11-6, Nb11-72, Nb11-75.

Example 3 Antibody Affinity Measurement

The binding kinetics of the 14-week blocking TSLP nanoantibodies were detected through bio-layer interferometry (BLI). For kinetic measurements, the candidate antibody was diluted to 5ug/mL in PBST buffer and the TSLP-Fc antigen was diluted in a 2-fold gradient with 6 concentration gradients (starting at 20 nM and diluted in 2-fold gradients) with PBST buffer, instrument operating conditions 30 ℃, Shake speed 1000rpm was set. Antibodies were captured for 60 seconds using a probe coated with Protein A. Binds to gradient diluted antigen with a binding time of 240 seconds; dissociation time is 300 seconds; Regeneration was performed twice with 10 mM glycine (pH 1.7) for 5 seconds each time. It was analyzed with Fortebio Analysis version 9.0, fitted with Global mode, and the association rate (Kon), dissociation rate (Kdis), and dissociation constant KD were calculated. The results are as shown in FIG. 2 .

Example 4 Expression of humanized bivalent antibodies in Pichia pastoris

Humanization improvement is performed on the candidate antibody to keep the variable region unchanged, humanization design is performed on the four frame region sequences, and the method of improvement is referred to the method of Example 4 of patent CN2018101517526. The amino acid sequences of the antibodies after humanization are shown in Table 1.

The humanized antibody was constructed in a bivalent form, linked with a linker (G ₄ S) 4, and the linked sequences were as shown in SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42, followed by Pichia pastoris. and expressed. Briefly, the expression method is as follows. (1) The TSLP nanoantibody divalent sequence was constructed as a pPICZaA vector. (2) After linearization with Sac I restriction endonuclease, X-33 soluble cells were electrotransfected. (3) The electrotransformed samples were respectively coated on YPD plate medium containing different concentrations of bleomycin and cultured in a culture tank at 30° C. for 3-4 days. (4) After single clones grow in plate medium, the single clones on the plates of different concentrations are placed in BMGY medium, and when the OD value of the BMGY culture medium reaches about 20, the cells are collected and replaced with BMMY medium, 28 ° C., 250 rpm cultured in. (5) Thereafter, samples are taken every 24 hours, and samples are collected by adding methanol with a final volume of 1%, centrifuged at 12,000 rpm for 5 minutes, and the supernatant is collected at -20 , and induction was continued for 5 days, and then the culture was terminated. (6) SDS-PAGE detection was performed on the obtained supernatant sample. Shake flask expression levels of bivalent single domain antibodies are shown in FIG. 3 .

Example 5 Detection of blocking activity of humanized bivalent antibodies by ELISA

The blocking activity of the purified bivalent humanized antibody was detected by ELISA. The human TSLP antigen protein was divided into 96-well plates and 4 incubated overnight. Wash 5 times with PBST and add 300uL 1% BSA blocking solution to 37 incubated for 2 hours. After washing 5 times with PBST, 50uL of gradient diluted antibody sample was added and 50uL of 0.02ug/mL biotinylated TSLPR protein was added to each well, 37 incubated for 1 hour. After washing 5 times with PBST, 100uL of SA-HRP (1:100000 dilution) was added, and 37 incubated for 1 hour. After washing 5 times with PBST, 100 uL of TMB coloring solution was added, and 37 After developing the color for 10 minutes, the reaction was terminated by adding 50 uL/well of 2M H ₂ SO ₄ , and the absorption value at a wavelength of 450 nm was measured using a microplate reader. The results are shown in Figure 4, and the blocking activity of the humanized bivalent antibody after 3 weeks was significantly superior to that of the control antibody Tezepelumab.

Example 6 Inhibitory effect of humanized bivalent antibody on pSTAT5 signaling pathway in BaF3/TSLPR-IL7R cells

The well-grown BaF3/TSLPR-IL7R cells were centrifuged at 1000 rpm for 5 minutes, the cells were resuspended in PBS, and 1×10 ⁵ cells per well were divided into 96-well plates. 25uL of diluted human TSLP factor was mixed with a gradient diluted bivalent humanized antibody or Tezepelumab, incubated at 37°C for 30 minutes, and then the mixture was put into cells and left for 20 minutes at 37°C and 5% CO ₂ . . After washing the cells with PBS, formaldehyde was added, and pre-chilled methanol was added and incubated for 10 minutes. Finally, anti-phosphorylated STAT5-PE-labeled antibody was added and incubated for 30 minutes, and after washing the cells, the PE signal was detected by flow cytometry. The results are shown in Figure 5, and the inhibitory effect of the humanized antibody for 2 weeks on the pSTAT5 signaling pathway in BaF3/TSLPR-IL7R cells was significantly superior to that of the control antibody Tezepelumab.

Example 7 Evaluation of yield of humanized bivalent antibody in 7L fermentation tank

The glycerol bacteria, which are the expression strains of the three antibodies, were amplified and cultured as first-class seeds at a ratio of 1:100, and then transferred to a fresh medium to perform second-class seed culture. After waiting until the second-class seed culture was passed, 7L It was placed in a fermentation tank and fermentation culture was performed, and ammonia water was automatically added to adjust the pH of the fermentation culture to 6.0. During the culture process, samples were taken regularly to detect the pH of the fermentation broth, the wet weight of the cells, and the OD value of the bacterial solution, and the stirring rotation speed, tank pressure, and oxygen ventilation were adjusted according to the change in the amount of dissolved oxygen. A glycerol-fed medium and a methanol-fed medium were added at different stages of the fermentation culture according to changes in the wet weight and dissolved oxygen content of the fermented cells. Fermentation was stopped after induction culture with methanol for 160 h to 200 h. As a result of the detection, the yields of the three humanized bivalent antibodies were 17 g/L, 19 g/L, and 23 g/L, respectively. The antibody production has reached the highest level in the industry, far higher than the reported production of all types of antibody fermentation and expression.

Sequence information:

SEQ ID NO.1 :

GFTLDDSDMG

SEQ ID NO.2 :

ISSLGGT

SEQ ID NO.3 :

APGTDRYSDCPNEYSV

SEQ ID NO.4 :

QVQLQESGGGSVQAGGSLRLSCTAS

SEQ ID NO.5 :

WYRQAPGDECELVST

SEQ ID NO.6:

YYADSVKGRFTISHDNAKNTVYLQMNSLKPHDTAVYYC

SEQ ID NO.7 :

WGQGTQVTVSS

SEQ ID NO.8:

QVQLQESGGGSVQAGGSLRLSCTASGFTLDDSDMGWYRQAPGDECELVSTISSLGGTYYADSVKGRFTISHDNAKNTVYLQMNSLKPHDTAVYYCAPGTDRYSDCPNEYSVWGQGTQVTVSS

SEQ ID NO.9:

CAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTACAGCCTCTGGATTCACTTTGGATGATTCTGACATGGGCTGGTACCGCCAGGCTCCAGGGGATGAGTGCGAGTTGGTCTCAACTATTAGTAGTTTGGGTGGCACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCCATGACAACGCCAAGAACGGTGTATCTGCAAATGAA CAGCCTGAAACCTCACGACACGGCCGTGTATTACTGTGCGCCGGGGACAGACCGTTATAGCGACTGCCCTAATGAGTATAGCGTCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA

SEQ ID NO. 10:

EVQLLESGGGLVQPGGSLRLSCTAS

SEQ ID NO.11:

WGQGTLVTVSS

SEQ ID NO. 12:

EVQLLESGGGLVQPGGSLRLSCTASGFTLDDSDMGWYRQAPGDECELVSTISSLGGTYYADSVKGRFTISHDNAKNTVYLQMNSLKPHDTAVYYCAPGTDRYSDCPNEYSVWGQGTLVTVSS

SEQ ID NO. 13:

GAGGTTCAATTGTTGGAATCTGGTGGTGGTTTGGTTCAACCAGGTGGTTCTTTGAGATTGTCTTGTACTGCTTCTGGTTTCACCTTGGACGATTCTGATATGGGATGGTACAGACAAGCTCCAGGAGATGAGTGTGAGTTGGTTTCTACTATCTCTTCCTTGGGTGGAACCTACTACGCTGATTCTGTCAAGGGTCGTTTCACTATTTCTCACGATAATGCTAAGAACACCGTTTACTTGCA AATGAACTCTTTAAAGCCACATGATACTGCCGTTTACTACTGTGCTCTGGTACTGATAGATACTCTGACTGTCCAAACGAATACTCCGTTTGGGGTCAGGGTACTTTGGTTACTGTCTCTTCC

SEQ ID NO. 14:

GFTFDDSDMG

SEQ ID NO.15:

ISSDGMT

SEQ ID NO. 16:

AATKYSSDYDVAEDWRRGVCGDMDY

SEQ ID NO. 17:

QVQLQESGGGSVQAGETLRLSCTAS

SEQ ID NO. 18:

WYRQAPGNECELVSI

SEQ ID NO. 19:

YYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAVYYC

SEQ ID NO. 20:

WGKGTQVTVSS

SEQ ID NO. 21:

QVQLQESGGGSVQAGETLRLSCTASGFTFDDSDMGWYRQAPGNECELVSIISSDGMTYYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAVYYCAATKYSSDYDVAEDWRRGVCGDMDYWGKGTQVTVSS

SEQ ID NO. 22:

CAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTCGGTGCAGGCTGGAGAGACTCTGAGACTCTCCTGTACAGCCTCTGGATTCACTTTTGATGATTCTGACATGGGCTGGTACCGCCAGGCTCCAGGGAATGAGTGCGAGTTGGTCTCAATTATTAGTAGTGATGGTATGACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCCAAGACAACGCCAAGAACACGGTGTATCTGCAAAT GAACAGCCTGAAACCTGAGGACACAGCCGTGTATTACTGTGCGGCGACGAAGTACTCCAGCGACTATGACGTAGCTGAGGATTGGAGACGCGGGGTCTGTGGAGACATGGACTACTGGGGCAAAGGAACCCAGGTCACCGTCTCCTCA

SEQ ID NO.23:

EVQLLESGGGLVQPGETLRLSCTAS

SEQ ID NO. 24:

WGKGTLVTVSS

SEQ ID NO.25:

EVQLLESGGGLVQPGETLRLSCTASGFTFDDSDMGWYRQAPGNECELVSIISSDGMTYYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAVYYCAATKYSSDYDVAEDWRRGVCGDMDYWGKGTLVTVSS

SEQ ID NO. 26:

GAAGTCCAATTGCTTGAATCCGGTGGTGGATTAGTTCAACCAGGTGAGACCTTGAGACTGTCCTGTACCGCTTCCGGTTTCACTTTCGACGACTCCGACATGGGTTGGTACAGACAGGCTCCAGGTAATGAGTTGAGTTGGTTTCTATTATTTCCTCTGATGGTATGACTTACTACGCTGATTCTGTTAAGGGTAGATTCACTATCTCTCAAGACAATGCTAAGAACACTGTTTACTTGCAAAT GAACTCTTTGAAGCCTGAAGATACCGCCGTCTACTACTGTGCTGCCACCAAGTACTCCTCCGATTATGATGTCGCTGAAGATTGGAGAAGAGGAGTTTGTGGAGATATGGATTACTGGGGTAAAGGTACTTTGGTTACCGTTTCTTCT

SEQ ID NO. 27:

GFTSGGSDMG

SEQ ID NO. 28:

ISSDGST

SEQ ID NO. 29:

AATDYGLGPPPSSTGQCYGMDY

SEQ ID NO. 30:

QVQLQESGGGSVQAGGSLRLSCTAS

SEQ ID NO. 31:

WYRQAPGNECDLVSY

SEQ ID NO. 32:

YYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAVYYC

SEQ ID NO. 33:

WGKGTQVTVSS

SEQ ID NO. 34:

QVQLQESGGGSVQAGGSLRLSCTASGFTSGGSDMGWYRQAPGNECDLVSYISSDGSTYYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAVYYCAATDYGLGPPPSSTGQCYGMDYWGKGTQVTVSS

SEQ ID NO. 35:

CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTACAGCCTCTGGATTCACTTCTGGCGGTTCTGACATGGGCTGGTACCGCCAGGCTCCAGGGAATGAGTGCGACTTGGTCTCATATATTAGTAGTGATGGTAGCACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCCAAGACAACGCCAAGAACGGTGTATCTGCAAATGA ACAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTGCGGCCACCGACTATGGGTTGGGGCCGCCCCCTTCTTCGACGGGCCAATGTTACGGCATGGACTACTGGGGCAAAGGAACCCAGGTCACCGTCTCCTCA

SEQ ID NO. 36:

EVQLLESGGGLVQPGGSLRLSCTAS

SEQ ID NO. 37:

WGKGTLVTVSS

SEQ ID NO. 38:

EVQLLESGGGLVQPGGSLRLSCTASGFTSGGSDMGWYRQAPGNECDLVSYISSDGSTYYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAVYYCAATDYGLGPPPSSTGQCYGMDYWGKGTLVTVSS

SEQ ID NO. 39:

GAAGTTCAGTTGTTGGAATCAGGTGGTGGTTTGGTTCAACCAGGAGGTTCTCTGAGATTGTCTTGTACTGCTTCCGGTTTCACTTCCGGAGGTTCTGACATGGGATGGTACCGTCAAGCTCCTGGTAACGAGTGCGACTTGGTTTCTTACATCTCTTCCGACGGTTCCACTTACTACGCTGATTCTGTTAAGGGTAGATTCACTATTTTCTCAAGACAACGCTAAGAATACTGTTTACTTGC AGATGAACTCTTTGAAGCCAGAGGATACCGCTGTTACTATTGTGCCGCCACTGATTACGGTTTGGGTCCTCCACCATCTTCTACTGGACAATGTTACGGTATGGATTACTGGGGTAAAGGTACCCTGGTCACCGTTTCCTCT

SEQ ID NO.40:

EVQLLESGGGLVQPGGSLRLSCTASGFTLDDSDMGWYRQAPGDECELVSTISSLGGTYYADSVKGRFTISHDNAKNTVYLQMNSLKPHDTAVYYCAPGTDRYSDCPNEYSVWGQGTLVTVSSGGGGSGGGGSGGGGSGGGSEVQLLESGGGLVQPGGSLRLSCTASGFTLDDSDMGWYRQAPGDECELVSTISSLGGTYYA DSVKGRFTISHDNAKNTVYLQMNSLKPHDTAVYYCAPGTDRYSDCPNEYSVWGQGTLVTVSS

SEQ ID NO.41:

EVQLLESGGGLVQPGETLRLSCTASGFTFDDSDMGWYRQAPGNECELVSIISSDGMTYYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAVYYCAATKYSSDYDVAEDWRRGVCGDMDYWGKGTLVTVSSGGGGSGGGGSGGGGSGGGSEVQLLESGGGLVQPGETLRLSCTASGFTFDDSDMGWYRQAPGNECELVSI ISSDGMTYYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAVYYCAATKYSSDYDVAEDWRRGVCGDMDYWGKGTLVTVSS

SEQ ID NO.42:

EVQLLESGGGLVQPGGSLRLSCTASGFTSGGSDMGWYRQAPGNECDLVSYISSDGSTYYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAVYYCAATDYGLGPPPSSTGQCYGMDYWGKGTLVTVSSGGGGSGGGGSGGGGSGGGSEVQLLESGGGLVQPGGSLRLSCTASGFTSGGSDMGWYRQAPGNECDLVSYISS DGSTYYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAVYYCAATDYGLGPPPSSTGQCYGMDYWGKGTLVTVSS

All documents cited herein are incorporated herein by reference as if a document were separately incorporated by reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and such equivalent forms also fall within the scope defined by the claims of the present invention. .

Sequence listing <110> SHANGHAI NOVAMAB BIOPHARMACEUTICALS CO., LTD. <120> Anti-TSLP nanobodies and their applications <130> P2022-0980 <150> CN202210111727.1 <151> 2022-01-26 <160> 42 <170> PatentIn version 3.5 <210> 1 <211> 10 <212 > PRT <213> artificial sequence <220> <223> CDR1 <400> 1 Gly Phe Thr Leu Asp Asp Ser Asp Met Gly 1 5 10 <210> 2 <211> 7 <212> PRT <213> artificial sequence <220 > <223> CDR2 <400> 2 Ile Ser Ser Leu Gly Gly Thr 1 5 <210> 3 <211> 16 <212> PRT <213> artificial sequence <220> <223> CDR3 <400> 3 Ala Pro Gly Thr Asp Arg Tyr Ser Asp Cys Pro Asn Glu Tyr Ser Val 1 5 10 15 <210> 4 <211> 25 <212> PRT <213> artificial sequence <220> <223> FR1 <400> 4 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser 20 25 <210> 5 <211> 15 <212> PRT <213> artificial sequence <220> <223> FR2 < 400> 5 Trp Tyr Arg Gln Ala Pro Gly Asp Glu Cys Glu Leu Val Ser Thr 1 5 10 15 <210> 6 <211> 38 <212> PRT <213> artificial sequence <220> <223> FR3 <400> 6 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser His Asp Asn 1 5 10 15 Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro His Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 7 <211> 11 <212> PRT <213> artificial sequence <220> <223> FR4 <400> 7 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 8 <211> 122 <212> PRT <213> artificial sequence <220> <223> the VHH chain of the nanobody <400> 8 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Leu Asp Asp Ser 20 25 30 Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Asp Glu Cys Glu Leu Val 35 40 45 Ser Thr Ile Ser Ser Leu Gly Gly Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser His Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro His Asp Thr Ala Val Tyr Cys Ala 85 90 95 Pro Gly Thr Asp Arg Tyr Ser Asp Cys Pro Asn Glu Tyr Ser Val Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 9 <211> 366 <212> DNA <213> artificial sequence <220> <223> the polynucleotide sequence <400> 9 caggtgcagc tgcaggagtc tggaggaggc tcggtgcagg ctggagggtc tctgagactc 60 tcctgtacag cctctggatt cactttggat gattctgaca tgggctggta ccgccaggct 120 ccaggggatg agtgcgagtt ggtctcaact attagtagtt tgggtggcac atactatgca 180 gactccgtga agggcc gatt caccatctcc catgacaacg ccaagaacac ggtgtatctg 240 caaatgaaca gcctgaaacc tcacgacg gccgtgtatt actgtgcgcc ggggacagac 300 cgttatagcg actgccctaa tgagtatagc gtctggggcc aggggaccca ggtcaccgtc 360 tcctca 366 <2 10> 10 <211> 25 <212> PRT <213> artificial sequence <220> <223> FR1 <400> 10 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser 20 25 <210> 11 <211> 11 <212> PRT <213> artificial sequence <220> <223> FR4 <400> 11 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 12 <211> 122 <212> PRT <213> artificial sequence <220> <223> the VHH chain of the nanobody <400> 12 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Leu Asp Asp Ser 20 25 30 Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Asp Glu Cys Glu Leu Val 35 40 45 Ser Thr Ile Ser Ser Leu Gly Gly Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser His Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro His Asp Thr Ala Val Tyr Cys Ala 85 90 95 Pro Gly Thr Asp Arg Tyr Ser Asp Cys Pro Asn Glu Tyr Ser Val Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 13 <211> 366 <212> DNA <213> artificial sequence <220> <223> the polynucleotide sequence < 400 > 13 gaggttcaat tgttggaatc tggtggtggt ttggttcaac caggtggttc tttgagattg 60 tcttgtactg cttctggttt caccttggac gattctgata tgggatggta cagacaagct 120 ccaggagatg agtgtgagtt ggtttctact atctct tcct tgggtggaac ctactacgct 180 gattctgtca agggtcgttt cactatttct cacgataatg ctaagaacac cgtttacttg 240 caaatgaact ctttaaagcc acatgatact gccgtttact actgtgctcc tggtactgat 300 agatactctg actgtccaaa cgaatactcc gtttggggtc agg gtacttt ggttactgtc 360 tcttcc 366 <210> 14 <211> 10 <212> PRT <213> artificial sequence <220> <223> CDR1 <400> 14 Gly Phe Thr Phe Asp Asp Ser Asp Met Gly 1 5 10 <210> 15 <211> 7 <212> PRT <213> artificial sequence <220> <223> CDR2 <400> 15 Ile Ser Ser Asp Gly Met Thr 1 5 <210> 16 <211> 25 <212> PRT <213> artificial sequence <220> <223> CDR3 < 400> 16 Ala Ala Thr Lys Tyr Ser Ser Asp Tyr Asp Val Ala Glu Asp Trp Arg 1 5 10 15 Arg Gly Val Cys Gly Asp Met Asp Tyr 20 25 <210> 17 <211> 25 <212> PRT <213> artificial sequence <220> <223> FR1 <400> 17 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Glu 1 5 10 15 Thr Leu Arg Leu Ser Cys Thr Ala Ser 20 25 <210> 18 <211> 15 <212> PRT <213> artificial sequence <220> <223> FR2 <400> 18 Trp Tyr Arg Gln Ala Pro Gly Asn Glu Cys Glu Leu Val Ser Ile 1 5 10 15 <210> 19 <211> 38 <212 > PRT <213> artificial sequence <220> <223> FR3 <400> 19 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn 1 5 10 15 Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 20 <211> 11 <212> PRT <213> artificial sequence <220> <223> FR4 <400> 20 Trp Gly Lys Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 21 <211> 131 <212> PRT <213> artificial sequence <220> <223> the VHH chain of the nanobody <400> 21 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Glu 1 5 10 15 Thr Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Asp Asp Ser 20 25 30 Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Asn Glu Cys Glu Leu Val 35 40 45 Ser Ile Ile Ser Ser Asp Gly Met Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Ala Thr Lys Tyr Ser Ser Asp Tyr Asp Val Ala Glu Asp Trp Arg Arg 100 105 110 Gly Val Cys Gly Asp Met Asp Tyr Trp Gly Lys Gly Thr Gln Val Thr 115 120 125 Val Ser Ser 130 <210> 22 <211> 393 <212> DNA <213> artificial sequence <220> <223> the polynucleotide sequence <400> ctggta ccgccaggct 120 ccagggaatg agtgcgagtt ggtctcaatt attagtagtg atggtatgac atactatgca 180 gactccgtga agggccgatt caccatctcc caagacaacg ccaagaacac ggtgtatctg 240 caaatgaaca gcctgaaacc tgaggacaca gccgtgtatt actgtgcggc gacgaagtac 300 tccagcgact atgacgtagc tgaggattgg agacgcgggg tctgtggaga catggactac 360 tggggcaaag gaacccaggt ca ccgtctcc tca 393 <210> 23 <211> 25 <212> PRT <213> artificial sequence <220> <223> FR1 <400 > 23 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Glu 1 5 10 15 Thr Leu Arg Leu Ser Cys Thr Ala Ser 20 25 <210> 24 <211> 11 <212> PRT <213> artificial sequence <220> <223> FR4 <400> 24 Trp Gly Lys Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 25 <211> 131 <212> PRT <213> artificial sequence <220> <223> the VHH chain of the nanobody <400> 25 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Glu 1 5 10 15 Thr Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Asp Asp Ser 20 25 30 Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Asn Glu Cys Glu Leu Val 35 40 45 Ser Ile Ile Ser Ser Asp Gly Met Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Ala Thr Lys Tyr Ser Ser Asp Tyr Asp Val Ala Glu Asp Trp Arg Arg 100 105 110 Gly Val Cys Gly Asp Met Asp Tyr Trp Gly Lys Gly Thr Leu Val Thr 115 120 125 Val Ser Ser 130 <210> 26 <211> 393 <212> DNA <213> artificial sequence <220> <223> the polynucleotide sequence <400> 26 gaagtccaat tgcttgaatc cggtggtgga ttagttcaac caggtgagac cttgagactg 60 tcctgtaccg cttccggttt cactttcgac gactccgaca tgggttggta cagacaggct 120 ccaggtaatg agtgtgagtt ggtttctatt atttcctctg atggtatgac ttactacgct 180 gattctgtta agggtagatt cactatctct caagaca atg ctaagaacac tgtttacttg 240 caaatgaact ctttgaagcc tgaagatacc gccgtctact actgtgctgc caccaagtac 300 tcctccgatt atgatgtcgc tgaagattgg agaagaggag tttgtggaga tatggattac 360 tggggtaaag gtactttggt taccgtttct tct 393 <210> 27 <211> 10 <212> PRT <213> artificial sequence <220> <223> CDR1 <400> 27 Gly Phe Thr Ser Gly Gly Ser Asp Met Gly 1 5 10 <210> 28 <211> 7 <212> PRT <213> artificial sequence <220> <223> CDR2 <400> 28 Ile Ser Ser Asp Gly Ser Thr 1 5 <210> 29 <211> 22 <212> PRT <213> artificial sequence <220> <223> CDR3 <400> 29 Ala Ala Thr Asp Tyr Gly Leu Gly Pro Pro Ser Ser Thr Gly Gln 1 5 10 15 Cys Tyr Gly Met Asp Tyr 20 <210> 30 <211> 25 <212> PRT <213> artificial sequence <220> <223> FR1 <400> 30 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser 20 25 <210> 31 <211> 15 <212> PRT <213> artificial sequence <220> <223> FR2 <400> 31 Trp Tyr Arg Gln Ala Pro Gly Asn Glu Cys Asp Leu Val Ser Tyr 1 5 10 15 <210> 32 <211> 38 <212> PRT <213> artificial sequence < 220> <223> FR3 <400> 32 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn 1 5 10 15 Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 33 <211> 11 <212> PRT <213> artificial sequence <220> <223> FR4 <400> 33 Trp Gly Lys Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210 > 34 <211> 128 <212> PRT <213> artificial sequence <220> <223> the VHH chain of the nanobody <400> 34 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Ser Gly Gly Ser 20 25 30 Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Asn Glu Cys Asp Leu Val 35 40 45 Ser Tyr Ile Ser Ser Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Ala Thr Asp Tyr Gly Leu Gly Pro Pro Pro Ser Ser Thr Gly Gln Cys 100 105 110 Tyr Gly Met Asp Tyr Trp Gly Lys Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 35 <211> 384 <212> DNA <213> artificial sequence <220> <223> the polynucleotide sequence <400> 35 caggtgcagc tgcaggagtc tgggggaggc tcggtgcagg ctggagggtc tctgagactc 60 tcctgtacag cctctggatt cacttctggc ggttctgaca tgggctggta ccgccaggct 120 ccagggaatg a gtgcgactt ggtctcatat attagtagtg atggtagcac atactatgca 180 gactccgtga agggccgatt caccatctcc caagacaacg ccaagaacac ggtgtatctg 240 caaatgaaca gcctgaaacc tgaggacacg gccgtgtatt actgtgcggc caccgactat 300 gggttggggc cgcccccttc ttcgacgggc caatgttacg gcatggacta ctggggcaaa 360 ggaacccagg tcaccgtctc ctca 384 <210> 36 <211> 25 <212> PRT <213> artificial sequence <220> <223> FR1 <400> 36 Glu Val Gln Le u Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser 20 25 <210> 37 <211> 11 <212> PRT <213> artificial sequence <220> <223> FR2 <400> 37 Trp Gly Lys Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 38 <211> 128 <212> PRT <213> artificial sequence <220> <223> the VHH chain of the nanobody <400> 38 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Ser Gly Gly Ser 20 25 30 Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Asn Glu Cys Asp Leu Val 35 40 45 Ser Tyr Ile Ser Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Cys Ala 85 90 95 Ala Thr Asp Tyr Gly Leu Gly Pro Pro Ser Ser Thr Gly Gln Cys 100 105 110 Tyr Gly Met Asp Tyr Trp Gly Lys Gly Thr Leu Val Thr Val Ser Ser 115 120 125 < 210> 39 <211> 384 <212> DNA <213> artificial sequence <220> <223> the polynucleotide sequence <400> 39 gaagttcagt tgttggaatc aggtggtggt ttggttcaac caggaggttc tctgagattg 60 tcttgtactg cttccggttt cacttccgga gg ttctgaca tgggatggta ccgtcaagct 120 cctggtaacg agtgcgactt ggtttcttac atctcttccg acggttccac ttactacgct 180 gg taccctgg tcaccgtttc ctct 384 <210> 40 <211> 264 <212> PRT <213> artificial sequence <220> <223> the sequences after ligation <400> 40 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Leu Asp Asp Ser 20 25 30 Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Asp Glu Cys Glu Leu Val 35 40 45 Ser Thr Ile Ser Ser Leu Gly Gly Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser His Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro His Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Pro Gly Thr Asp Arg Tyr Ser Asp Cys Pro Asn Glu Tyr Ser Val Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Leu Asp Asp Ser Asp Met 165 170 175 Gly Trp Tyr Arg Gln Ala Pro Gly Asp Glu Cys Glu Leu Val Ser Thr 180 185 190 Ile Ser Ser Leu Gly Gly Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg 195 200 205 Phe Thr Ile Ser His Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met 210 215 220 Asn Ser Leu Lys Pro His Asp Thr Ala Val Tyr Tyr Cys Ala Pro Gly 225 230 235 240 Thr Asp Arg Tyr Ser Asp Cys Pro Asn Glu Tyr Ser Val Trp Gly Gln 245 250 255 Gly Thr Leu Val Thr Val Ser Ser 260 <210> 41 <211> 282 <212> PRT <213> artificial sequence <220> <223> the sequences after ligation <400> 41 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Glu 1 5 10 15 Thr Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Asp Asp Ser 20 25 30 Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Asn Glu Cys Glu Leu Val 35 40 45 Ser Ile Ile Ser Ser Asp Gly Met Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Cys Ala 85 90 95 Ala Thr Lys Tyr Ser Ser Asp Tyr Asp Val Ala Glu Asp Trp Arg Arg 100 105 110 Gly Val Cys Gly Asp Met Asp Tyr Trp Gly Lys Gly Thr Leu Val Thr 115 120 125 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 130 135 140 Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly 145 150 155 160 Gly Leu Val Gln Pro Gly Glu Thr Leu Arg Leu Ser Cys Thr Ala Ser 165 170 175 Gly Phe Thr Phe Asp Asp Ser Asp Met Gly Trp Tyr Arg Gln Ala Pro 180 185 190 Gly Asn Glu Cys Glu Leu Val Ser Ile Ile Ser Ser Asp Gly Met Thr 195 200 205 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn 210 215 220 Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp 225 230 235 240 Thr Ala Val Tyr Tyr Cys Ala Ala Thr Lys Tyr Ser Ser Asp Tyr Asp 245 250 255 Val Ala Glu Asp Trp Arg Arg Gly Val Cys Gly Asp Met Asp Tyr Trp 260 265 270 Gly Lys Gly Thr Leu Val Thr Val Ser Ser 275 280 <210> 42 <211> 276 <212> PRT <213> artificial sequence <220 > <223> the sequences after ligation <400> 42 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Ser Gly Gly Ser 20 25 30 Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Asn Glu Cys Asp Leu Val 35 40 45 Ser Tyr Ile Ser Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Ala Thr Asp Tyr Gly Leu Gly Pro Pro Ser Ser Thr Gly Gln Cys 100 105 110 Tyr Gly Met Asp Tyr Trp Gly Lys Gly Thr Leu Val Thr Val Ser Ser 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val 145 150 155 160 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr 165 170 175 Ser Gly Gly Ser Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Asn Glu 180 185 190 Cys Asp Leu Val Ser Tyr Ile Ser Ser Asp Gly Ser Thr Tyr Tyr Ala 195 200 205 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn 210 215 220 Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val 225 230 235 240 Tyr Tyr Cys Ala Ala Thr Asp Tyr Gly Leu Gly Pro Pro Pro Ser Ser 245 250 255 Thr Gly Gln Cys Tyr Gly Met Asp Tyr Trp Gly Lys Gly Thr Leu Val 260 265 270Thr Val Ser Ser 275

Claims (19)

As an anti-TSLP nanoantibody,
The complementarity determining region (CDR) of the VHH chain in the nanoantibody,
(1) CDR1 represented by SEQ ID NO: 1, CDR2 represented by SEQ ID NO: 2, and CDR3 represented by SEQ ID NO: 3;
(2) CDR1 represented by SEQ ID NO: 14, CDR2 represented by SEQ ID NO: 15, and CDR3 represented by SEQ ID NO: 16; and
(3) CDR1 represented by SEQ ID NO: 27, CDR2 represented by SEQ ID NO: 28, and CDR3 represented by SEQ ID NO: 29, characterized in that at least one selected from the group consisting of -TSLP nanoantibodies. According to claim 1,
The VHH chain of the anti-TSLP nanoantibody further comprises a frame region (FR), the frame region (FR),
(1) FR1 represented by SEQ ID NO: 4, FR2 represented by SEQ ID NO: 5, FR3 represented by SEQ ID NO: 6, and FR4 represented by SEQ ID NO: 7;
(2) FR1 represented by SEQ ID NO: 10, FR2 represented by SEQ ID NO: 5, FR3 represented by SEQ ID NO: 6, and FR4 represented by SEQ ID NO: 11;
(3) FR1 represented by SEQ ID NO: 17, FR2 represented by SEQ ID NO: 18, FR3 represented by SEQ ID NO: 19, and FR4 represented by SEQ ID NO: 20;
(4) FR1 represented by SEQ ID NO: 23, FR2 represented by SEQ ID NO: 18, FR3 represented by SEQ ID NO: 19, and FR4 represented by SEQ ID NO: 24;
(5) FR1 represented by SEQ ID NO: 30, FR2 represented by SEQ ID NO: 31, FR3 represented by SEQ ID NO: 32, and FR4 represented by SEQ ID NO: 33; and
(6) Anti-TSLP characterized in that at least one selected from the group consisting of FR1 represented by SEQ ID NO: 36, FR2 represented by SEQ ID NO: 31, FR3 represented by SEQ ID NO: 32, and FR4 represented by SEQ ID NO: 37 nano antibody. According to claim 1,
The amino acid sequence of the VHH chain of the anti-TSLP nanoantibody is selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 34, SEQ ID NO: 38, or a combination thereof anti-TSLP nanoantibodies that do. As an anti-TSLP antibody,
An anti-TSLP antibody, which is an antibody against a TSLP epitope and has the anti-TSLP nanoantibody according to claim 1. As an anti-TSLP antibody,
The anti-TSLP antibody, characterized in that the antibody comprises at least one anti-TSLP nano-antibody according to claim 1. According to claim 4 or 5,
The anti-TSLP antibody, characterized in that the antibody comprises a monomeric, bivalent antibody, and / or multivalent antibody. As a polynucleotide,
The polynucleotide is characterized in that it encodes a protein selected from the group consisting of the anti-TSLP nanoantibody according to claim 1, the anti-TSLP antibody according to claim 4, or the anti-TSLP antibody according to claim 5 . As an expression vector,
An expression vector characterized in that the expression vector contains the polynucleotide according to claim 7. As a host cell,
The host cell is characterized by containing the expression vector according to claim 8 or having the polynucleotide according to claim 7 integrated into its genome. As a method for generating anti-TSLP nanoantibodies,
(a) obtaining a culture containing the anti-TSLP nanoantibody by culturing the host cell according to claim 9 under conditions suitable for producing the nanoantibody;
(b) isolating or recovering the anti-TSLP nanoantibody from the culture; and
(c) optionally purifying and/or modifying the anti-TSLP nanoantibody obtained in step (b); As an immunoconjugate,
The immunoconjugate,
(a) the anti-TSLP nanoantibody according to claim 1, the anti-TSLP antibody according to claim 4, or the anti-TSLP antibody according to claim 5; and
(b) contains a junction moiety selected from the group consisting of detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, gold nanoparticles/nanorods, magnetic nanoparticles, viral coat proteins or VLPs, or combinations thereof. Immunoconjugate, characterized in that. As a multispecific antibody,
The multispecific antibody comprises the anti-TSLP nano-antibody according to claim 1, the anti-TSLP antibody according to claim 4, or the anti-TSLP antibody according to claim 5. As a recombinant protein,
The recombinant protein,
(i) the anti-TSLP nanoantibody according to claim 1, the anti-TSLP antibody according to claim 4, or the anti-TSLP antibody according to claim 5; and
(ii) an optional tag sequence to aid expression and/or purification; As a pharmaceutical composition,
The pharmaceutical composition,
(i) the anti-TSLP nanoantibody according to claim 1, the anti-TSLP antibody according to claim 4 or the anti-TSLP antibody according to claim 5, or the immunoconjugate according to claim 11 or the multispecificity according to claim 12 an antibody or recombinant protein according to claim 13; and
(ii) a pharmaceutically acceptable carrier; a pharmaceutical composition characterized in that it contains. The anti-TSLP nanoantibody according to claim 1, the anti-TSLP antibody according to claim 4 or the anti-TSLP antibody according to claim 5, the immunoconjugate according to claim 11 or the multispecific antibody according to claim 12, or the anti-TSLP antibody according to claim 13. As a use of the recombinant protein according to claim 14 or the pharmaceutical composition according to claim 14,
(a) manufactures drugs for the prevention and/or treatment of TSLP-related disorders; and/or (b) manufacturing a TSLP detection reagent, detection plate or kit. As a method for detecting TSLP protein in a sample,
The method,
(1) The anti-TSLP nanoantibody according to claim 1, the anti-TSLP antibody according to claim 4, or the anti-TSLP antibody according to claim 5, or the immunoconjugate according to claim 11 or claim 12 contacting the multispecific antibody or the recombinant protein according to claim 13; and
(2) detecting whether an antigen-antibody complex is formed, indicating that the TSLP protein is present in the sample when the complex is formed; As a TSLP protein detection reagent,
The detection reagent,
(i) the anti-TSLP nanoantibody according to claim 1, the anti-TSLP antibody according to claim 4 or the anti-TSLP antibody according to claim 5, or the immunoconjugate according to claim 11 or the multispecificity according to claim 12 an antibody or recombinant protein according to claim 13; and
(ii) a detectably acceptable carrier; TSLP protein detection reagent comprising a. As a kit for detecting TSLP protein,
The kit for detecting TSLP protein, characterized in that the kit contains the immunoconjugate according to claim 7 or the detection reagent and instructions according to claim 17. As a use of the immunoconjugate according to claim 11,
A use characterized by preparing a contrast agent for detecting TSLP protein in the body.